It has been suggested that the small fraction of tumor cells capable of initiating local or distant recurrence exhibit properties of stem cells, and that identification and molecular characterization of putative cancer stem cells will lead to better outcomes through targeted cancer stem cell therapies in appropriately selected patients. Clinical studies examining the predictive and prognostic significance of these cells have been limited by several factors, including variable methods for isolating single cells from tissues, the need for multiple markers to distinguish committed from undifferentiated cells, and the availability of fresh tissue from patients for study. Originating from the primary tumor, circulating tumor cells (in the blood) and disseminated tumor cells (in the bone marrow) are attractive cancer stem cell candidates, and practical clinical surrogates for cancer stem cell study because they exist as single cells, and can be quantified using available FDA approved technology. Therefore, we propose a translational strategy to examine primary, circulating, and disseminated tumor cells from breast cancer patients to test our central hypothesis that circulating and disseminated tumor cells present in the blood and bone marrow of non-metastatic breast cancer patients represent cancer stem cells which are precursors to distant metastases and potential targets for novel treatment. Our hypothesis is based on preliminary data from an ongoing IRB-approved clinical protocol that demonstrates disseminated tumor cells in the bone marrow of patients with non-metastatic breast cancer are positive for the expression of cancer stem cell markers of tumor initiating capacity; CD44+CD24- and aldehyde dehydrogenase-1 (ALDH1). Translational aims are based on three IRB protocols and tailored to the material collected, treatment approach, and patient population in each trial. The translational work represents an established multidisciplinary collaboration between Massimo Cristofanilli, MD Breast Medical Oncology, James Reuben, PhD, Hematopathology, and Wendy Woodward, MD-PhD, Clinical and Experimental Breast Radiation Oncology. The goals are 1) Measure CTCs and DTCs in patients without metastatic disease, assess these cells for stem cell features and correlate these to outcome and ALDH1 staining in the primary; 2) Measure CTCs and primary tumor stem cells in patients receiving neoadjuvant chemotherapy and correlate response to therapy between these measures as well as transplant and culture primary biopsy material for molecular studies; 3) Determine key mediators of -catenin and Notch-1 crosstalk in radioresistance of cancer stem cells, and test inhibition of these pathways as radiosensitizers. In these studies we hope to demonstrate that CTCs/DTCs are prognostic and predictive surrogates for cancer stem cells that are easily accessible for examination on the single cell level. This approach could be used to select patients for individualized therapy using targeted sensitizing agents based on the proposed preclinical studies.